To facilitate understanding of the composition and method of this invention, the following definitions of terms used throughout this specification and claims are provided.                1. The term “equivalent(s)” is used in the chemical sense to describe a quantity of a specific component of a molecule or of a specific ion of a salt. Thus a 1M solution of GdCl3 would contain 1 equivalent of the gadolinium ion and 3 equivalents of the chloride ion in 1 liter of solution.        2. The term “light” is used to designate any form of electromagnetic radiation, including the ultraviolet, visible, and infrared region of the spectrum.        3. The term “photon” is used to designate an individual particle of light.        4. The term “lanthanide” is used to designate any of the lanthanide elements (atomic number 57-71) as well as the lanthanide-like yttrium and the actinide elements (atomic number 89-103).        5. The term “chemical compound” follows the common usage of the field of chemistry and is used to describe any pure substance that is formed when atoms of two or more different elements combine and create a new material that has a constant composition throughout and properties that are unlike those of the constituent elements.        6. The term “complex” is used to describe any species in which a metal atom or ion is bonded to an organic moiety. Examples of complexes include the lanthanide(III) macrocycles of U.S. Pat. Nos. 5,373,093 and 5,696,240; the cryptates of U.S. Pat. Nos. 4,927,923, 5,162,508, and 5,534,622; the phthalamidyl-containing chelates of U.S. Pat. No. 6,515,113; the salicylamidyl-containing chelates of U.S. Pat. No. 6,406,297; and the chelates formed with the reaction product of diethylenetriaminepentaacetic acid dianhydride (DTPAA) and p-aminosalicylic acid of U.S. Pat. No. 4,962,045. In such a complex, each bond between the metal atom or ion and the organic moiety consists of a shared electron pair originally belonging solely to the organic moiety. Since many complexes can be isolated or identified by standard techniques, they are chemical compounds.        7. The term “ligand” is used to describe the organic moiety of a complex.        8. The term “unidentate ligand” is used to describe a molecule or ion that binds to a metal atom or ion through a single site, and more specifically through a single atom or a through the electrons of one multiple bond between a pair of atoms.        9. The term “multidentate ligand” is used to describe a molecule or ion that can bind to a metal atom or ion through two or more sites, and more specifically through two or more atoms, or through the electrons of two or more multiple bonds between pairs of atoms, or through a combination of thereof.        10. The term “eteroatom” is used to indicate any atom of a cyclic molecule or of a cyclic portion of a molecule or ion, that is not carbon.        11. The term “macrocycle” is used to describe a cyclic organic compound in which the cycle consists of nine or more members, including carbon and all eteroatoms with three or more of such atoms capable of acting as electron pair donors (ligands) toward metal atoms or ions.        12. The term “macrocyclic ligand” is used to describe a macrocycle that functions as ligand in a complex.        13. The term “lanthanide macrocycle” means a complex where one or more lanthanide atoms or ions are bound into the cavity of a macrocyclic ligand.        14. The abbreviation “LnMac” will be used to describe all of the functionalized macrocycles taught in U.S. Pat. Nos. 5,373,093 and 5,696,240.        15. The registered trademark “Quantum Dye®” is and has been used to describe all of the functionalized macrocycles taught in U.S. Pat. Nos. 5,373,093 and 5,696,240.        16. The term “unitary solution” is used to describe a homogenous solution that consists of a single phase.        17. The term “unitary luminescence enhancing solution” is used to describe a unitary solution that after evaporation of the solvent to dryness results in a solid that enhances the luminescence of an energy transfer acceptor lanthanide(III) complex by a mechanism other than completing the complexation of the lanthanide ion.        18. The term “homogeneous” is used to describe a material that has a constant composition except for the material to be measured.        19. The term “homogeneous solid composition” is used to describe a material that does not flow, contains two or more chemical species, and is homogenous.        20. The term “luminescence enhancing solid composition” is the homogeneous solid composition produced by the drying of the unitary luminescence enhancing solution.        21. The term “processed specimen” is used to describe the material present after conventional clinical or research processing.        22. The term “labeled specimen containing composition” is used to describe a composition that includes a processed specimen with one or more labels embedded in the luminescence enhancing solid composition.        23. The terms “nucleic acid material” and “nucleic acids” each refer to deoxyribonucleotides, ribonucleotides, or analogues thereof in either single- or double-stranded form. Unless specifically limited, the term encompasses nucleic acids containing known analogues of natural nucleotides that have similar properties as the reference natural or antisense nucleic acid. Thus “nucleic acids” includes but is not limited to DNA, cDNA, RNA, antisense RNA, double-stranded RNA, and oligonucleotides. A therapeutic nucleic acid can comprise a nucleotide sequence encoding a therapeutic gene product, including a polypeptide or an oligonucleotide.        Nucleic acids can further comprise a gene (e.g., a therapeutic gene), or a genetic construct (e.g., a gene therapy vector). The term “gene” refers broadly to any segment of DNA associated with a biological function. A gene encompasses sequences including but not limited to a coding sequence, a promoter region, a cis-regulatory sequence, a non-expressed DNA segment that is a specific recognition sequence for regulatory proteins, a non-expressed DNA segment that contributes to gene expression, a DNA segment designed to have desired parameters, or combinations thereof. A gene can be obtained by a variety of methods, including cloning from a biological sample, synthesis based on known or predicted sequence information, and recombinant derivation of an existing sequence.        24. The term “energy transfer donor” is used to designate any molecular or ionic species capable of transferring all or part of the energy absorbed from light to another molecular or ionic species.        25. The term “energy transfer donor metal ion” is used to designate any metal ion that is itself, or is part of a complex that serves as, an “energy transfer donor”. This complex need not itself be ionic. This energy transfer can be direct or indirect if the presence of the metal ion induces another molecule or ion, including but not limited to those with which it has formed a complex, to transfer energy to another molecular or ionic species.        26. The term “energy transfer donor complex of a metal ion” is used to designate any complex of an energy transfer donor metal ion that is capable of transferring all or part of the energy absorbed from light to another molecular or ionic species.        27. The term “energy transfer acceptor” is used to designate any molecular or ionic species capable of accepting all or part of the energy absorbed from light by another molecular or ionic species.        28. The term “energy transfer acceptor lanthanide(III)” is used to designate a lanthanide(III) that accepts energy from one or more molecules or ions, and as a result of this process emits part of this energy as a photon.        29. The term “energy transfer acceptor lanthanide(III) complex” is used to designate any lanthanide(III) complex or compound that is capable of accepting energy, directly or indirectly, from an energy transfer donor and of emitting photons with energies equal to or less than the energy received.        30. The term “resonance energy transfer” is used to describe a process by which an energy transfer donor transfers energy to an energy transfer acceptor.        31. The term “RET” is an abbreviation for “resonance energy transfer”.        32. The term “heterogeneous resonance energy transfer” is used to describe a process by which an energy transfer donor transfers all or part of the energy absorbed from light to an energy transfer acceptor, where the energy transfer donor and energy transfer acceptor are different molecular species.        33. The term “homogeneous resonance energy transfer” is used to describe a process by which any molecular species transfers all or part of the energy absorbed from light to another member of the same molecular species.        34. The term “columinescence” is used to describe the increase in luminescence brought about by the addition of a luminescence-enhancing amount of at least one energy transfer donor complex of a metal ion to one or more energy transfer acceptor lanthanide(III) complexes, where the emission spectrum of the energy transfer donor species differs from that of the one or more energy transfer acceptor lanthanide(III) complexes. The “term Lanthanide Enhanced Luminescence” that has been previously used is a synonym for columinescence        35. The term “columinescence solution” is used to describe a solution, the use of which results in “columinescence.” The term “lanthanide enhanced luminescence solution” that has been previously used is a synonym for columinescence        36. The term “LEL solution” is an abbreviation for “lanthanide enhanced luminescence solution.”        37. The term “reactive functionality” is used to mean a first atom or group capable of reacting with a second atom or group forming a covalent bond with it, as previously used in U.S. Pat. Nos. 5,373,093 and 5,696,240 to mean that both the first and second atom or group are capable of forming a covalent bond with one another. These atoms or groups include but are not limited to amine, azide, alcoholic hydroxyl, phenolic hydroxyl, aldehyde, carboxylic acid, carboxamide, halogen, isocyanate, isothiocyanate, mercapto and nitrile substituents. Functionalized alkyl, functionalized aryl-substituted alkyl, functionalized aryl, and functionalized alkyl-substituted aryl signify the respective alkyl, aryl-substituted alkyl, aryl, and alkyl-substituted aryl groups substituted with a reactive functionality.        38. A “peptide” is a polymer that is composed of monomer units that primarily are amino acids. The peptide monomer units are linked to one another by amide bonds.        39. The term “label” means the species or moiety that permits a molecule to be detected or to be affected non-destructively by a physical means.        40. The term “tag” is a synonym for “label”.        41. The term “optical-label” means the species or moiety that permits a molecule to be detected by optical means including emission of photons from both singlet and triplet electronic excited states.        42. The term “labeled” designates a molecule that has formed a covalent bond with a label.        43. The term “tagged” is a synonym for “labeled”.        44. The term “labeled-polymer” means a polymer to which one or more labels are attached.        45. The term “tagged-polymer” is a synonym for “labeled-polymer”.        46. The term “labeled-polymer-conjugate” means a labeled-polymer where this polymer has formed a covalent bond with a molecular species other than itself or its label(s).        47. The term “tagged-polymer-conjugate” is a synonym for is a synonym for “Labeled-polymer-conjugate”.        48. The term “fluorescence” means a process by which an electron of a molecule or ion that is in an electronic singlet state (a state in which the spins of all electrons are paired) absorbs the energy contained in one or more photons, with the result that this electron is elevated to a higher energy singlet state, and subsequently an electron of this molecule or ion loses energy in the form of a quantum of energy and deactivates to a lower energy state. This process does not involve a change in the electronic spin multiplicity of the molecule or ion. This quantum of energy can be in the form of an emission of a photon or transfer of energy to a neighboring molecule or ion.        49. The term “fluorophore” means a molecule or ion capable of fluorescence.        50. The term “luminescence” means all processes by which an electron of a molecule or ion absorbs the energy contained in one or more photons, with the result that this electron is elevated to a higher energy singlet state, subsequently relaxes to a lower energy triplet state, and subsequently energy is lost from an electron of this molecule or ion in the form of a quantum of energy with the concurrent deactivation of this electron to a lower state. This process involve a change of the electronic spin multiplicity of the molecule or ion. This quantum of energy can be in the form of an emission of a photon or transfer of energy to a neighboring molecule or ion.        51. The term “lumiphore” means a molecule or ion capable of luminescence.        52. The term “light absorption” means a process by which an electron in a molecule or ion absorbs the energy contained in one or more photons.        53. The term “optical-label” means a label capable of fluorescence, luminescence, or absorption.        54. The term “luminescence-label” means an optical-label that is capable of luminescence, such as a lanthanide macrocycle.        55. “The term fluorescence-label” means an optical-label that is capable of fluorescence.        56. “The term absorption-label” means an optical-label that is capable of absorption.        57. The term “specific combining pair” means a pair of molecules that forms a stable complex.        58. The term “bridging molecule” means any molecule that can be simultaneously bound to a label and a member of a specific combining pair.        59. The term “analyte” means any compound of interest, naturally occurring or synthetic, that can bind to a member of a specific combining pair that is to be quantitated.        60. An “analyte-binding species” is the member of a specific combining pair that can form a stable complex with an analyte. These analyte-binding species include but are not limited to:                    a) an antibody or antibody fragment.                            (i) Such antibodies or fragments may be defined to include polyclonal antibodies from any native source and native or recombinant monoclonal antibodies of classes IgG, IgM, IgA, IgD, and IgE; hybrid derivatives, and fragments of antibodies including Fab, Fab′ and F(ab′)2; humanized or human antibodies; recombinant or synthetic constructs containing the complementarity determining regions of an antibody, and the like. The methods useful for construction of all such antibodies are known to those of skill in the art.                                    b) a polynucleotide, polynucleotide fragment, or an oligonucleotide.                            (i) Such polynucleotides, polynucleotide fragments, or oligonucleotides include but are not limited to: deoxynucleic acids, DNAs; ribonucleic acids, RNAs; and peptide nucleic acids, PNAs.                                    c) a protein that is a member of specific combining pair.                            (i) Such proteins include but are not limited to avidin, streptavidin, and their derivatives.                                    d) a lectin.                        61. The term “indirectly labeled” means a process where an analyte-binding species is a member of two specific combining pairs. The other member of the first specific combining pair is a labeled molecule. The other member of the second specific combining pair is an analyte. The analyte-binding species is bound to both the analyte and the labeled molecule.        62. The term “co-hybridization” means a process where two DNA samples differing in at least one property are hybridized with a third DNA.        63. The term “material” is defined to include: cells, organisms, bacteria, viruses, histological sections, organic and inorganic particulates and matter, and any other discernible material which provides diagnostic and/or analytical information whatsoever.        64. The term “microscopic analysis” is defined to be a process wherein a microscope under human and/or a machine control is used for visualization, analysis, and/or enumeration, and/or categorization, and/or photography, and/or electronic image acquisition of material.        65. The term “receiving surface member” will be used in a generic sense to describe all discrete objects which serve as substrates to support material for microscopic viewing and/or observation and/or analysis. The current, most common receiving surface member is a microscope slide, which is a glass rectangular object that is approximately 1 mm thick, 25 mm wide, and 75 mm long. These are the items conventionally referred to as microscope slides for laboratory and commercial purposes.        
As used herein and in the appended statements of the invention, the singular forms “a”, “and”, and “the” include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to “a construct” includes a plurality of such constructs, and so forth.
The term “about”, as used herein when referring to a measurable value such as an amount of weight, time, dose, etc. is meant to encompass variations of in one embodiment ±20% or ±10%, in another embodiment ±5%, in another embodiment ±1%, and in still another embodiment ±0.1% from the specified amount, as such variations are appropriate to perform the disclosed methods.
The term “expression”, as used herein to describe a genetic construct, generally refers to the cellular processes by which a biologically active polypeptide or biologically active oligonucleotide is produced from a DNA sequence.
The term “construct”, as used herein to describe a genetic construct, refers to a composition comprising a vector used for gene therapy or other application. In one embodiment, the composition also includes nucleic acids comprising a nucleotide sequence encoding a therapeutic gene product, for example a therapeutic polypeptide or a therapeutic oligonucleotide. In one embodiment, the nucleotide sequence is operatively inserted with the vector, such that the nucleotide sequence encoding the therapeutic gene product is expressed. The term “construct” also encompasses a gene therapy vector in the absence of a nucleotide sequence encoding a therapeutic polypeptide or a therapeutic oligonucleotide, referred to herein as an “empty construct.” The term “construct” further encompasses any nucleic acid that is intended for in vivo studies, such as nucleic acids used for triplex and antisense pharmacokinetic studies.
The terms “bind”, “binding”, “binding activity” and “binding affinity” are believed to have well-understood meanings in the art. To facilitate explanation of the present invention, the terms “bind” and “binding” are meant to refer to protein-protein interactions that are recognized to play a role in many biological processes, such as the binding between an antibody and an antigen, and between complementary strands of nucleic acids (e.g. DNA-DNA, DNA-RNA, and RNA-RNA). Exemplary protein-protein interactions include, but are not limited to, covalent interactions between side chains, such as disulfide bridges between cysteine residues; hydrophobic interactions between side chains; and hydrogen bonding between side chains.
The terms “binding activity” and “binding affinity” are also meant to refer to the tendency of one protein or polypeptide to bind or not to bind to another protein or polypeptide. The energetics of protein-protein interactions are significant in “binding activity” and “binding affinity” because they define the necessary concentrations of interacting partners, the rates at which these partners are capable of associating, and the relative concentrations of bound and free proteins in a solution. The binding of a ligand to a target molecule can be considered specific if the binding affinity is about 1×104 M−1 to about 1×106 M−1 or greater.
The phrase “specifically (or selectively) binds”, for example when referring to the binding capacity of an antibody, also refers to a binding reaction which is determinative of the presence of the antigen in a heterogeneous population of proteins and other biological materials. The phrase “specifically (or selectively) binds” also refers to selective targeting of a targeting molecule, such as the hybridization of a RNA molecule to a nucleic acid of interest under a set of hybridization conditions as disclosed herein below.
Table of AbbreviationsBSAbovine serum albumincDNAcomplementary DNACIAchloroform/isoamyl alcoholCTABcetyltrimethylammonium bromidedATPdeoxyadenosine triphosphatedCTPdeoxycytidine triphosphatedGTPdeoxyguanosine triphosphateDMFdimethylformamideDMSOdimethylsulfoxideDNAdeoxyribonucleic aciddNTPdeoxynucleotide triphosphatedTTPdeoxythymidine triphosphatedUTP2′-deoxyuridine 5′-TriphosphateEDTAethylenediaminetetraacetic acidEqvequivalent(s)Eqv/Lequivalent(s) per LiterEuMac-d-UTPEuMac-5-deoxyuridine triphosphateh or hrhour(s)H2PDCA2,6-pyridinedicarboxylic acidHTTFAthenoyltrifluoroacetoneLliter(s)LELLanthanide Enhanced LuminescenceLn(III) or Ln3+a trivalent lanthanide cationLnMacSee Definition 14.LnMac-d-UTPLnMac-5-deoxyuridine triphosphateMmolarMmolarmgmilligram(s)minminute(s)mLmilliliter(s)mMmillimolarmmmillimetermmolmillimole(s)mmol/Lmillimoles per litermRNAmessenger RNAmsecmillisecond(s)nEqvnanoequivalentngnanogram(s)nmolnanomole(s)PBphosphate bufferPBSphosphate buffered salinePCRpolymerase chain reactionPDCAThe dianion of 2,6-pyridinedicarboxylic acidPEGpolyethylene glycolRETResonance Energy TransferRNAribonucleic acidrpmrevolutions per minuteSDSsodium dodecyl sulfatesecseconds(s)SmMac-d-UTPSmMac-5-deoxyuridine triphosphateTbMac-d-UTPTbMac-5-deoxyuridine triphosphateTTFAThe anion of thenoyltrifluoroacetoneμgmicrogram(s)μLmicroliter(s)μMmicromolarμmolmicromole(s)μmol/Lmicromoles per literμsecmicrosecond(s)